Cassava leaves as an alternative protein source: Effect of alkaline parameters and precipitation conditions on protein extraction and recovery.

Alternative protein sources have been required to meet the significant plant protein demand. Agro-industrial by-products such as leaves have considerable potential as a source of macromolecules once they are mostly discarded as waste. The current study evaluated dried cassava leaves as a protein source. First, alkaline extraction parameters (solid-liquid ratio, pH, and temperature) were optimized and the run that result in the highest protein yield were acidified at pH 2.5 or 4. The influence of carbohydrate solubilized on protein precipitation was also evaluated by removing it via alcoholic extraction prior to precipitation. The experimental design showed that high pH and temperature conditions associated with a low solid-liquid ratio led to increased protein yields. The presence of carbohydrates in the supernatant significantly influenced protein precipitation. The protein concentrate had around 17.51% protein when it was obtained from a supernatant with carbohydrates, while protein content increased to 26.88% when it was obtained from carbohydrate-free supernatant. The precipitation pH also influenced protein content, whereas protein content significantly decreased when pH increased from 2.5 to 4. The natural interaction between carbohydrates and proteins from cassava leaves positively influenced the emulsion stability index and the foaming capacity and stability. Thus, the presented results bring insights into challenges in extracting and precipitation proteins from agro-industrial by-products.

Extraction of Proteins from Fermented Food.

The metaproteomic approach allows a deep microbiome characterization in different complex systems. Based on metaproteome data, microbial communities' composition, succession, and functional role in different environmental conditions can be established.The main challenge in metaproteomic studies is protein extraction, and although many protocols have been developed, a few are focused on the protein extraction of fermented foods. In this chapter, a reproducible and efficient method for the extraction of proteins from a traditionally fermented starchy food is described. The method can be applied to any fermented food and aims to enrich the extraction of proteins from microorganisms for their subsequent characterization.

Improved MALDI-TOF MS Identification of Mycobacterium tuberculosis by Use of an Enhanced Cell Disruption Protocol.

Mycobacterium tuberculosis is the microorganism that causes tuberculosis, a disease affecting millions of people worldwide. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a fast, reliable, and cost-effective method for microorganism identification which has been used for the identification of Mycobacterium spp. isolates. However, the mycobacteria cell wall is rich in lipids, which makes it difficult to obtain proteins for MALDI-TOF MS analysis. In this study, two cell preparation protocols were compared: the MycoEx, recommended by MALDI-TOF instrument manufacturer Bruker Daltonics, and the MycoLyser protocol described herein, which used the MagNA Lyser instrument to enhance cell disruption with ethanol. Cell disruption and protein extraction steps with the two protocols were performed using the Mycobacterium tuberculosis H37Rv strain, and the MALDI-TOF MS results were compared. The MycoLyser protocol allowed for improved Biotyper identification of M. tuberculosis since the log(score) values obtained with this protocol were mostly ≥ 1.800 and significantly higher than that underwent MycoEx processing. The identification reliability was increased as well, considering the Bruker criteria. In view of these results, it is concluded that the MycoLyser protocol for mycobacterial cell disruption and protein extraction improves the MALDI-TOF MS method's efficacy for M. tuberculosis identification.

Isolation and Characterization of Protein Fractions for Valorization of Sacha Inchi Oil Press-Cake.

The growing interest in plant-based food protein sources has provided opportunities for the valorization of agri-food by-products, driving the food industry towards more sustainable development. In this study, three extraction procedures (varying the pH value (7.0 and 11.0) and the addition of salt (0 and 5%)) were investigated to obtain seven different protein fractions (SIPF) from Sacha Inchi oil press-cake (SIPC), which were characterized in terms of their protein content, electrophoretic profile, secondary structure, and techno-functional properties. Extractions at pH 11.0 without salt addition produced the highest values of protein content, extraction yield, protein recovery, and protein concentration increase (84.0%, 24.7%, 36.5%, and 1.5-fold, respectively). Under these extraction conditions, the electrophoretic analysis indicated that most of the SIPC proteins were extracted. SIPF displayed an excellent oil absorption capacity (4.3-9.0 w/w), and interesting foam activity (36.4-133.3%). The solubility and emulsifying activity of the albumin fractions were significantly higher than those of the other fractions (~87 vs. <15.8%, and 280-370 vs. <140 m2/g, respectively). Correlation analysis showed that the secondary structure of the SIPF significantly influences their techno-functional properties. These results indicate that SIPC is a by-product of great potential for protein extraction processes, and as a valorization strategy for technical cycle solutions for the Sacha Inchi productive chain in the circular economy context.

Edible insect as an alternative protein source: a review on the chemistry and functionalities of proteins under different processing methods.

The consumption of edible insects can be anadvantageous alternative to the conventional food supply chain, which involves global water waste, land deficit, undernutrition, and starvation. Besides the nutritional aspects, insect proteins have demonstrated a wide range of functional properties such as foamability, emulsifying and gelling abilities. The protein content and amino acid profile of some insects have revealed a good nutritional value and interesting functional properties. However, it is crucial to comprehend how the protein quality is affected by insect feeding, drying, and defatting. There is a knowledge gap about the impact of industrial treatment, such as pH, ionic strength, and heat treatment, on insect proteins' functional properties. In this review, we have aimed to highlight the potential application of insect proteins as a nutritional source and their promising technological applications. The study reported the principal insect protein characterization methodologies that have been investigated in the literature aiming to correlate the physicochemical parameters to possible protein functionalities. The research on the functional properties of insect proteins is at the exploratory level. Further detailed studies are needed to clarify the structure-function relation of insect proteins and how these functionalities and insect processing can increase consumer acceptance.

Nutritional Quality, Techno-Functional Characteristics, and Safety of Biomass Powder and Protein Isolate Produced from Penicillium maximae.

This study investigated the suitability of Penicillium maximae biomass powder and protein isolate as a food product or food ingredient. The biomass powder is rich in proteins (34.8%) and insoluble fiber (36.2%) but poor in lipids (3.1%). Strong water hydration (8.3 g/g, 8.5 g/g) and oil holding (6.9 g/g, 16.3 g/g) capacity were observed in the biomass powder and protein isolate, respectively, besides 100% emulsion stability, indicating multiple applications in the food industry. No locomotor impairment was induced in Drosophila melanogaster flies after consuming extracts of P. maximae biomass powder. Furthermore, decreased production of reactive oxygen species and preservation of survival, viability, and fertility parameters were observed in the nematode Caenorhabditis elegans, which reinforces the potential of P. maximae biomass for human and animal consumption. Together, the results show the vast food applicability of P. maximae biomass and protein isolate as protein substitutes with several health and environmental benefits.

Structural and Physicochemical Characterization of Extracted Proteins Fractions from Chickpea (Cicer arietinum L.) as a Potential Food Ingredient to Replace Ovalbumin in Foams and Emulsions.

Chickpeas are the third most abundant legume crop worldwide, having a high protein content (14.9-24.6%) with interesting technological properties, thus representing a sustainable alternative to animal proteins. In this study, the surface and structural properties of total (TE) and sequential (ALB, GLO, and GLU) protein fractions isolated from defatted chickpea flour were evaluated and compared with an animal protein, ovalbumin (OVO). Differences in their physicochemical properties were evidenced when comparing TE with ALB, GLO, and GLU fractions. In addition, using a simple and low-cost extraction method it was obtained a high protein yield (82 ± 4%) with a significant content of essential and hydrophobic amino acids. Chickpea proteins presented improved interfacial and surface behavior compared to OVO, where GLO showed the most significant effects, correlated with its secondary structure and associated with its flexibility and higher surface hydrophobicity. Therefore, chickpea proteins have improved surface properties compared to OVO, evidencing their potential use as foam and/or emulsion stabilizers in food formulations for the replacement of animal proteins.

Physico-chemical and colloidal properties of protein extracted from black soldier fly (Hermetia illucens) larvae.

The black soldier fly larvae (BSFL), Hermetia illucens (Linnaeus), has been largely utilized for animal feed. Due to its interesting composition, BSFL has great potential to be further implemented in the human diet. Herein we compared the flour and protein extract composition based on their moisture, ash, amino acids, mineral, and protein content. To have wide knowledge on protein profile and behavior, SDS-page electrophoresis, Fourier-transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) were used to give information about protein structure and thermal stability, respectively. The flour and protein extract contained respectively 37.3% and 61.1% of protein. DSC graph reported a glass transition temperature around 30 °C, recognizable by a shift in the curve, and an endothermic peak for solid melting at around 200 °C. FTIR analysis showed the main amide bands (A, B, I, II, III) for the flour and protein extract. The foam properties of BSFL protein extract were explored under different temperatures treatment, and the best foam stability was reached at 85 °C with 15 min of treatment. The data highlight the promising techno-functional properties of BSFL protein extract, and that the nutritional composition might be suitable for further use of BSFL as food fortification system.

Hevea brasiliensis latex proteomics: a review of analytical methods and the way forward.

Natural rubber or latex from the Hevea brasiliensis is an important commodity in various economic sectors in today's modern society. Proteins have been detected in latex since the early twentieth century, and they are known to regulate various biological pathways within the H. brasiliensis trees such as the natural rubber biosynthesis, defence against pathogens, wound healing, and stress tolerance. However, the exact mechanisms of the pathways are still not clear. Proteomic analyses on latex have found various proteins and revealed how they fit into the mechanisms of the biological pathways. In the past three decades, there has been rapid latex protein identification due to the improvement of latex protein extraction methods, as well as the emergence of two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). In this manuscript, we reviewed the methods of latex protein extraction that keeps on improving over the past three decades as well as the results of numerous latex protein identification and quantitation.

Effect of Ultrasound Application on Protein Yield and Fate of Alkaloids during Lupin Alkaline Extraction Process.

The objective of this work is to elucidate the fate of quinolizidine alkaloids (QA) during the lupin protein extraction process assisted with ultrasound and the evaluation of the nutritional and functional properties of the protein fraction. Proximal characterization, concentration of anti-nutritional compounds, amino acid profile and protein solubility profile of flours from three lupin species were (L. albus, L. angustifolius and L. mutabilis) assessed. The result showed a significant difference (p < 0.05) in protein concentration, fat, total alkaloids and particle size between the three species flours. Based on these parameters, the most different Lupinus species (L. mutabilis and L. angustifolius) were chosen to study the behavior of the protein fraction in terms of functionality, composition and resistance to thermal treatments. The results obtained for L. mutabilis described the ultrasound effect as beneficial for protein yield (14% more than control), QA reduction from bagasse (81% less than control) and protein isolate production (50% less than control). On the other hand, L. angustifolius was more resistant to the ultrasound effect with no significant difference between treatments (10 and 15 min) and control but with the lower toxicity and better amino acid score. These results will be useful to design processes to assist in the objective of meeting the future protein demand of the population.

Assessment of protein extraction and digestion efficiency of well-established shotgun protocols for heart proteomics.

Ischemic heart disease is the leading cause of deaths worldwide. Thus, understanding the molecular mechanisms underlying disease progression is needed. Due to heart importance and lack of studies evaluating different sample preparation methods for heart proteomics, we compared three well-established protocols in shotgun proteomics using dimethyl label quantitation to allow relative quantitation. The tested methods for the analysis of left ventricle (LV) tissue were: i) in-solution digestion (ISD); ii) on-pellet digestion (OPD); and iii) on-filter digestion (OFD). Protein extraction was done using SDS-containing buffer for OPD and OFD while this step was under urea-containing buffer for ISD. We used an optimized one-step reaction for reduction of disulfide bonds and alkylation of thiol groups in ISD and OPD. Using the same amount of tissue, we observed that OFD and ISD extracted significantly higher amount of protein than OPD. ISD outperformed OFD and OPD in the number of proteins identified. We did not observe significant bias related to physicochemical features of the identified proteins when comparing the three protocols. ISD was more efficient to identify low abundant proteins and yielded more proteins per protocol duration. Thus, we concluded that the optimized ISD suited better for heart proteomics than OFD and OPD.

A Rapid Extraction Method for mammalian cell cultures, suitable for quantitative immunoblotting analysis of proteins, including phosphorylated GCN2 and eIF2α.

Many studies require the detection and relative quantitation of proteins from cell culture samples using immunoblotting. Limiting factors are the cost of protease inhibitors, the time required to break cells and generate samples, as well as the high risk of protein loss during cell breakage procedures. In addition, a common problem is the viscosity of lysed samples due to the released genomic DNA. As a consequence, the DNA needs to be broken down prior to denaturing polyacrylamide protein gel electrophoresis (SDS-PAGE), e.g. by passing the sample through a syringe gauge needle, sonication, or DNase treatment. In a quest to find a more cost-effective, fast, and yet robust procedure, we found that cell lysis, protein denaturation, and DNA fragmentation can be done in only two steps: harvesting followed by a simple non-laborious 2nd step. Similarly to many pre-existing cell breakage procedures, in our Rapid Protein Extraction (RPE) method, proteins liberated from cells are immediately exposed to a denaturing environment. However, advantages of our method are: •No breaking buffer is needed, instead proteins are liberated directly into the denaturing protein loading buffer used for SDS-PAGE. Consequently, our RPE method does not require any expensive inhibitors.•The RPE method does not involve post-lysis centrifugation steps; instead all cell material is dissolved during the 2nd step, the mixing-heat-treatment step which is new to this method. This prevents potential protein loss that may occur during centrifugation. In addition, this 2nd step simultaneously shears the genomic DNA, making an additional step for DNA fragmentation unnecessary.•The generated samples are suitable for high-quality quantitative immunoblotting. With our RPE method we successfully quantified the phosphorylated forms of protein kinase GCN2 and its substrate eIF2α. In fact, the western signals were stronger and with less background, as compared to samples generated with a pre-existing method.

A Rapid and Reliable Method for Total Protein Extraction from Succulent Plants for Proteomic Analysis.

Crassulacean acid metabolism plants have some morphological features, such as succulent and reduced leaves, thick cuticles, and sunken stomata that help them prevent excessive water loss and irradiation. As molecular constituents of these morphological adaptations to xeric environments, succulent plants produce a set of specific compounds such as complex polysaccharides, pigments, waxes, and terpenoids, to name a few, in addition to uncharacterized proteases. Since all these compounds interfere with the analysis of proteins by electrophoretic techniques, preparation of high quality samples from these sources represents a real challenge. The absence of adequate protocols for protein extraction has restrained the study of this class of plants at the molecular level. Here, we present a rapid and reliable protocol that could be accomplished in 1 h and applied to a broad range of plants with reproducible results. We were able to obtain well-resolved SDS/PAGE protein patterns in extracts from different members of the subfamilies Agavoideae (Agave, Yucca, Manfreda, and Furcraea), Nolinoideae (Dasylirion and Beucarnea), and the Cactaceae family. This method is based on the differential solubility of contaminants and proteins in the presence of acetone and pH-altered solutions. We speculate about the role of saponins and high molecular weight carbohydrates to produce electrophoretic-compatible samples. A modification of the basic protocol allowed the analysis of samples by bidimensional electrophoresis (2DE) for proteomic analysis. Furostanol glycoside 26-O-ß-glucosidase (an enzyme involved in steroid saponin synthesis) was successfully identified by mass spectrometry analysis and de novo sequencing of a 2DE spot from an Agave attenuata sample.

Evaluation of cytotoxic activity of protein extracts from the leaves of Morinda pubescens on human cancer cell lines

ABSTRACT Biologically active proteins isolated from plant species can be used in traditional medicine as prolific resources for new drugs Morinda pubescens Sm., Rubiaceae, is a promising medicinal plant which is widely used in folk medicine to treat fever due to primary complex, ulcer and glandular swellings. In this study, proteins were extracted from the leaves of M. pubescens, and precipitated with ammonium sulphate at various saturation concentrations ranging from 20 to 80%. The precipitated protein sample obtained with 80% saturation was further purified using ultrafiltration membrane (<10 kDa). SDS-PAGE analysis identified the presence of crude and ultrafiltered protein bands. FTIR spectrum of the ultrafiltered protein fractions depicted the presence of hydroxyl and carbonyl groups of proteins. The ultrafiltered proteins exhibited increased cytotoxic activity on A549 cells at the concentrations ranging from 15 to 100 µg/ml. About 98% cell viability was also observed in Vero cells treated with the maximum concentration of 100 µg/ml of ultrafiltered protein extract. DNA fragmentation was observed in A549 cells treated with 10 µg/ml of ultrafiltered proteins, indicating the onset of apoptosis.

Potential application of microalga Spirulina platensis as a protein source.

The high protein level of various microalgal species is one of the main reasons to consider them an unconventional source of this compound. Spirulina platensis stands out for being one of the richest protein sources of microbial origin (460-630 g kg-1 , dry matter basis), having similar protein levels when compared to meat and soybeans. The use of S. platensis in food can bring benefits to human health owing to its chemical composition, since it has high levels of vitamins, minerals, phenolics, essential fatty acids, amino acids and pigments. Furthermore, the development of new protein sources to supply the shortage of this nutrient is an urgent need, and protein from S. platensis plays an important role in this scenario. In this sense, extraction processes that allow maximum protein yield and total utilization of biomass is an urgent need, and ultrasonic waves have proven to be an effective extraction technique. The number of scientific papers related to protein fraction from S. platensis is still limited; thus further studies on its functional and technological properties are needed. © 2016 Society of Chemical Industry.

Usefulness of matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry for identifying clinical Trichosporon isolates.

Trichosporon spp. have recently emerged as significant human pathogens. Identification of these species is important, both for epidemiological purposes and for therapeutic management, but conventional identification based on biochemical traits is hindered by the lack of updates to the species databases provided by the different commercial systems. In this study, 93 strains, or isolates, belonging to 16 Trichosporon species were subjected to both molecular identification using IGS1 gene sequencing and matrix-assisted laser desorption ionisation-time-of-flight (MALDI-TOF) analysis. Our results confirmed the limits of biochemical systems for identifying Trichosporon species, because only 27 (36%) of the isolates were correctly identified using them. Different protein extraction procedures were evaluated, revealing that incubation for 30 min with 70% formic acid yields the spectra with the highest scores. Among the six different reference spectra databases that were tested, a specific one composed of 18 reference strains plus seven clinical isolates allowed the correct identification of 67 of the 68 clinical isolates (98.5%). Although until recently it has been less widely applied to the basidiomycetous fungi, MALDI-TOF appears to be a valuable tool for identifying clinical Trichosporon isolates at the species level.

Sample preparation for arsenic speciation in terrestrial plants--a review.

Arsenic is an element widely present in nature. Additionally, it may be found as different species in several matrices and therefore it is one of the target elements in chemical speciation. Although the number of studies in terrestrial plants is low, compared to matrices such as fish or urine, this number is raising due to the fact that this type of matrix are closely related to the human food chain. In speciation analysis, sample preparation is a critical step and several extraction procedures present drawbacks. In this review, papers dealing with extraction procedures, analytical methods, and studies of species conservation in plants cultivated in terrestrial environment are critically discussed. Analytical procedures based on extractions using water or diluted acid solutions associated with HPLC-ICP-MS are good alternatives, owing to their versatility and sensitivity, even though less expensive strategies are shown as feasible choices.

A simple, economical and reproducible protein extraction protocol for proteomics studies of soybean roots.

Sample preparation is a critical step in two-dimensional gel electrophoresis (2-DE) of plant tissues. Here we describe a phenol/SDS procedure that, although greatly simplified, produced well-resolved and reproducible 2-DE profiles of protein extracts from soybean [Glycine max (L.) Merril] roots. Extractions were made in three replicates using both the original and simplified procedure. To evaluate the quality of the extracted proteins, ten spots were randomly selected and identified by mass spectrometry (MS). The 2-DE gels were equally well resolved, with no streaks or smears, and no significant differences were observed in protein yield, reproducibility, resolution or number of spots. Mass spectra of the ten selected spots were compared with database entries and allowed high-quality identification of proteins. The simplified protocol described here presents considerable savings of time and reagents without compromising the quality of 2-DE protein profiles and compatibility with MS analysis, and may facilitate the progress of proteomics studies of legume-rhizobia interactions.

Method optimization for proteomic analysis of soybean leaf: Improvements in identification of new and low-abundance proteins.

The most critical step in any proteomic study is protein extraction and sample preparation. Better solubilization increases the separation and resolution of gels, allowing identification of a higher number of proteins and more accurate quantitation of differences in gene expression. Despite the existence of published results for the optimization of proteomic analyses of soybean seeds, no comparable data are available for proteomic studies of soybean leaf tissue. In this work we have tested the effects of modification of a TCA-acetone method on the resolution of 2-DE gels of leaves and roots of soybean. Better focusing was obtained when both mercaptoethanol and dithiothreitol were used in the extraction buffer simultaneously. Increasing the number of washes of TCA precipitated protein with acetone, using a final wash with 80% ethanol and using sonication to ressuspend the pellet increased the number of detected proteins as well the resolution of the 2-DE gels. Using this approach we have constructed a soybean protein map. The major group of identified proteins corresponded to genes of unknown function. The second and third most abundant groups of proteins were composed of photosynthesis and metabolism related genes. The resulting protocol improved protein solubility and gel resolution allowing the identification of 122 soybean leaf proteins, 72 of which were not detected in other published soybean leaf 2-DE gel datasets, including a transcription factor and several signaling proteins.